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Journal of Virology, January 2001, p. 278-291, Vol. 75, No. 1
Department of
Microbiology1 and Department of
Comparative Medicine,4 University of Alabama,
Birmingham, Alabama 35294; Hematology-Oncology
Division2 and Department of
Microbiology,3 University of Pennsylvania,
Philadelphia, Pennsylvania 19104; AIDS Vaccine Program, SAIC
Frederick, NCI Frederick Cancer Research and Development Center,
Frederick, Maryland 217015;
Department of Surgery, Duke University Medical Center,
Durham, North Carolina 277106; and
MRC Laboratory for Molecular Cell Biology and Department of
Biochemistry, University College London, London, United
Kingdom7
Received 22 June 2000/Accepted 27 September 2000
Attenuated simian immunodeficiency viruses (SIVs) have been
described that produce low levels of plasma virion RNA and exhibit a
reduced capacity to cause disease. These viruses are particularly useful in identifying viral determinants of pathogenesis. In the present study, we show that mutation of a highly conserved tyrosine (Tyr)-containing motif (Yxx Infection by human immunodeficiency
viruses (HIVs) is characterized by persistent viral replication that in
most individuals in the absence of treatment leads to a progressive and
irreversible decline in the number of CD4+ lymphocytes and
eventually to AIDS (33). Although host immune responses
and genetic factors can influence the level of virus replication
(10, 12, 13, 77, 81), viral determinants undoubtedly play
key roles in affecting the development of disease (2, 25, 48,
60). The related simian immunodeficiency viruses (SIVs) have
provided powerful animal models to determine the contribution of viral
and host factors to disease progression (53, 92).
Interestingly, a number of SIVs that are markedly attenuated in vivo
have been derived. These viruses characteristically establish low to
undetectable plasma viral RNA levels and cause limited depletion of
CD4+ lymphocytes (3, 42). Although some
attenuated viruses have been reported to cause disease in neonatal
animals (4, 5) and in some adults after prolonged
infection (4, 5, 28, 89), they have been extremely useful
in identifying viral determinants for virulence in vivo. Moreover,
animals infected with attenuated SIVs have, in some cases, been
protected from infection and/or disease when challenged intravenously
(24) or mucosally (22, 44, 66, 72) with more
pathogenic homologous (24, 56, 79) or heterologous
(15, 103) strains. Thus, these viruses may provide
important clues for determining the nature of protective immunity for
SIV and HIV.
Mechanisms responsible for HIV and SIV attenuation in vivo could be due
to (i) intrinsic defects in virus infectivity and/or replicative
capacity, (ii) reduced cytopathic effects of viral infection on target
cells, and/or (iii) increased susceptibility of the virus to a host
immune response. Among the attenuated viruses studied to date, the best
characterized are those containing mutations in the viral
nef gene (42, 46). This gene encodes a 27-kDa myristoylated protein that has been associated with a number of biological effects, which include (i) downregulating CD4 and major histocompatibility complex class I molecules (1, 17, 19, 21, 37,
39, 59, 75, 85), (ii) augmenting virus infectivity (14,
26, 58, 67, 90), and (iii) altering T-cell signaling pathways
and/or development (6, 11, 37, 40, 83, 87, 100, 104).
Consequently, Nef could affect viral pathogenesis through multiple
mechanisms (29). In addition, other studies have
demonstrated that Nef-deleted viruses can be rendered more attenuated
by deletions of other accessory genes, such as vpr, vpx, and vif (30, 44). Another
well-characterized SIV variant, SIVmac1A11, exhibits a markedly
attenuated phenotype that appears to involve determinants in multiple
regions of the genome (63). Interestingly, given the
importance of the viral structural genes for infectivity, it is
remarkable that few examples exist in which specific mutations in these
genes are associated with reduced pathogenicity. This observation could
result from the high viral mutation rate, creating reversions or
compensatory mutations that can be selected over time to generate
progressively more fit viruses (16, 47, 94).
Alternatively, mutations in structural genes may be poorly tolerated
due to their deleterious effects on viral replication. Nonetheless, it
is reasonable to predict that mutations in important functional domains
of SIV and HIV structural proteins would have significant consequences
for pathogenesis and could, depending on their mechanism of action, be
combined with mutations in accessory genes in strategies to produce
viruses with reduced virulence.
In the present study we evaluated the in vivo effects of mutations in a
highly conserved tyrosine (Tyr)-dependent sorting motif in the SIV Env
cytoplasmic tail. For SIV and HIV, this Yxx Construction of recombinant viruses.
SIVmac239/nef-stop containing mutations in the Yxx
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.1.278-291.2001
In Vivo Attenuation of Simian Immunodeficiency
Virus by Disruption of a Tyrosine-Dependent Sorting Signal in the
Envelope Glycoprotein Cytoplasmic Tail
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) in the envelope glycoprotein (Env) cytoplasmic tail (amino acids YRPV at positions 721 to 724) can profoundly reduce the in vivo pathogenicity of SIVmac239. This domain
constitutes both a potent endocytosis signal that reduces Env
expression on infected cells and a sorting signal that directs Env
expression to the basolateral surface of polarized cells. Rhesus
macaques were inoculated with SIVmac239 control or SIVmac239 containing
either a Tyr-721-to-Ile mutation (SIVmac239Y/I) or a deletion of
Tyr-721 and the preceding glycine (
GY). To assess the in vivo
replication competence, all viruses contained a stop codon in
nef that has been shown to revert during in vivo but not in
vitro replication. All three control animals developed high viral loads
and disease. One of two animals that received SIVmac239Y/I and two of
three animals that received SIVmac239GY remained healthy for up to
140 weeks with low to undetectable plasma viral RNA levels and normal
CD4+ T-cell percentages. These animals exhibited ongoing
viral replication as determined by detection of viral sequences and
culturing of mutant viruses from peripheral blood mononuclear cells and
persistent anti-SIV antibody titers. In one animal that received
SIVmac239Y/I, the Ile reverted to a Tyr and was associated with a high
plasma RNA level and disease, while one animal that received
SIVmac239GY also developed a high viral load that was associated
with novel and possibly compensatory mutations in the TM cytoplasmic
domain. In all control and experimental animals, the nef
stop codon reverted to an open reading frame within the first 2 months
of inoculation, indicating that the mutant viruses had replicated well
enough to repair this mutation. These findings indicate that the Yxx signal plays an important role in SIV pathogenesis. Moreover, because
mutations in this motif may attenuate SIV through mechanisms that are
distinct from those caused by mutations in nef, this Tyr-based sorting signal represents a novel target for future models of
SIV and human immunodeficiency virus attenuation that could be useful
in new vaccine strategies.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
motif (where Y is a Tyr,
x is any amino acid, and is an amino acid with a bulky hydrophobic
side chain) (61, 69, 84) has been shown in vitro to
constitute both a potent endocytosis signal (8, 74, 78,
82) and a basolateral sorting signal (27, 54, 55).
These signals are analogous to those in cellular proteins that are
constitutively endocytosed from the plasma membrane, where binding of
the Yxx to µ2 chains of AP2 adapter complexes recruits cell
surface proteins into clathrin-coated pits (7, 8, 74).
Interactions with other adapter proteins, AP1 in particular, probably
underlie the ability of this motif to direct Env expression to the
basolateral surface of polarized cells (27, 54, 55). We
(82) and others (7, 27, 78) have demonstrated that this membrane-proximal motif can modulate the surface expression of Env on infected cells by recruiting Env glycoproteins that are not
incorporated into virions into clathrin-coated pits. We have proposed
that this motif could function in vivo to reduce the susceptibility of
infected cells to host immune responses (62).
Alternatively, the basolateral function of this signal could influence
pathogenesis by facilitating cell-to-cell infection and/or directing
the spread of virus to key anatomic areas (27). Here we
report that disruption of the membrane-proximal Yxx motif in the
pathogenic molecular clone SIVmac239 results in viruses that are able
to replicate in vitro but are markedly attenuated in their ability to
sustain high viral loads in vivo. These novel findings indicate the
importance of this highly conserved Env sorting signal in SIV (and
probably HIV) pathogenesis. Mutations in this signal could be useful in
complementary strategies to attenuate SIV and HIV through mechanisms
independent of those mediated by viral accessory genes.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
motif,
where Y is at amino acid position 721 of the SIVmac239 sequence, were
produced using pVP-1 and pVP-2 plasmids as previously described (50). The mutagenesis primers were
5'-TTAAGGCAGGGGATTAGGCCAGTGTTCC-3' for the Y721I mutation
and 5'-TTAAGGCAGAGGCCAGTGTTCTC-3' for the GY mutation
(Fig. 1). To produce viruses, pVP-1 and
pVP-2 were codigested with SphI, ligated with T4 ligase,
linearized with ApaI, and electroporated into CEMx174 cells.
Virus was harvested when cytopathic effects were observed, and the
stocks were quantified by measuring reverse transcriptase activity
(50).
View larger version (21K):
[in a new window]
FIG. 1.
Partial amino acid sequences of the membrane-spanning
and cytoplasmic domains of SIVmac239 and Tyr-721 mutants. Tyr-721 is
indicated by the arrow. Residues that are critical to the formation of
the endocytosis signal include Gly-720, Tyr-721, and Val-724 (8,
9).
Infection of macaques.
Eight juvenile rhesus macaques
(Macaca macaca), seronegative for antibodies to SIV, simian
T-cell leukemia virus, simian type D retroviruses, and herpesvirus type
B, were used in this study. Macaques were housed in isolation
facilities at the University of Alabama at Birmingham in accordance
with institutional and Animal Welfare Act guidelines. Before
inoculation of virus or collection of blood samples, the macaques were
anesthetized by intramuscular injection of ketamine hydrochloride (10 mg/kg), weighed, and examined for lymphadenopathy or other signs of
disease. The macaques were randomly placed in three groups and
inoculated intravenously with approximately 1,000 50% tissue culture
infective doses of either SIVmac239/nef-stop (three
macaques) or one of two SIVmac239/nef-stop env
mutant viruses: two macaques were inoculated with the Y721I cytoplasmic
tail mutant, and three macaques were inoculated with the GY mutant.
After virus inoculation, blood samples were collected from each animal
at regular intervals for hematologic analysis, to isolate and quantify
virus, and for serologic assays. The macaques were euthanized when
moribund, and complete necropsies were performed.
In vitro replication of virus. Macaque peripheral blood mononuclear cells (PBMC) were separated from whole blood by centrifugation through lymphocyte separation medium and then stimulated with concanavalin A at 106 cells/ml in RPMI 1640 medium containing antibiotics and 10% fetal bovine serum (10% medium). After 3 days, the PBMC were washed and aliquots of 107 cells were infected for 2 h with 1,000 to 3,000 50% tissue culture infective doses of the different virus strains before being washed and placed in 10% medium containing 8 U of interleukin-2/ml. Viral stocks were titrated by limiting dilution and infection of CEMx174 cells. Every 4 to 5 days, approximately 30% of the culture medium was removed, assayed for reverse transcriptase activity, and replaced with fresh medium.
Detection and quantification of virus. To isolate virus, macaque PBMC were cultured with phytohemagglutinin-stimulated normal human PBMC; culture supernatants were tested every 4 to 5 days for reverse transcriptase activity using standard procedures. The cultures were monitored 6 to 7 weeks before being identified as virus negative. Two assays were used to quantify plasma virion RNA: a branched-DNA (bDNA) signal amplification assay specific for SIV (performed at Chiron, now Bayer Corp.) was used with plasma containing heparin, and a subset of EDTA-treated plasma samples were assayed using a real-time reverse transcription-PCR (RT-PCR) assay (93). The results of the two assays were comparable.
Serum antibodies. Titers of antibodies specific for SIVmac239 were determined on serum samples after twofold serial dilutions using a highly cross-reactive HIV-2 enzyme immunoassay kit (Sanofi Pasteur Diagnostics, Seattle, Wash.). A CEMx174 cell-killing assay was used to determine neutralizing-antibody titers against either H9 cell line-adapted SIVmac251 or SIVmac239/nef-open grown in rhesus macaque PBMC (68).
Flow cytometry. To monitor changes in lymphocyte subsets, EDTA-treated blood samples were stained by a whole-blood lysis procedure with fluorochrome-labeled mouse anti-human monoclonal antibodies with high cross-reactivity to macaque cell surface antigens. All antibodies, reagents, and the FACS-STAR flow cytometer on which the analyses were performed were purchased from Becton Dickinson (Mountain View, Calif.). Lymphocytes were gated according to forward-scatter-versus-side-scatter characteristics, with negative cell populations excluded after identification with isotype-matched control antibodies.
PCR amplification and sequence analyses. Nested PCR of portions of the env TM and nef genes was performed with 1 µg of DNA from macaque PBMC obtained at various times after infection. Fragments of 393 and 310 bp, respectively, of the env TM were amplified with the following sets of primers: outer, 239env-I (5'-TAGAGGAGG-CACAAATTCAACAAG-3') (bases 8823 to 8846) and 239env-II (5'-TGCTGAATAGCCAA-GTCAAGAG-3') (bases 9215 to 9194); and inner, 239env-III (5'-GAATTACAAAAGTTG-AATAGCTGGG-3') (bases 8861 to 8885) and 239env-IV (5'-AATGAATATATTCTATCTG-CCAAGG-3') (bases 9170 to 9146). For the nef gene, fragments of 505 and 389 bp were amplified with the following sets of primers: outer, 239nef-I (5'-CTCTTAGGAGAGGT-GGAAGATGG-3') (bases 9423 to 9445) and 239nef-IV (5'-GAGCTGGATGCATTAAATAA-TGCTC-3') (bases 9927 to 9903); and inner, 239nef-II (5'-TTGAGCTCACTCTCTTGTGA-GGG-3') (bases 9480 to 9502) and 239nef-III (5'-GGGACTAATTTCCATAGCCAGCC-3') (bases 9868 to 9846). The positions of all primers are those of SIVmac239. PCR products were purified, cloned into the PCR vector (TA cloning kit; Invitrogen, San Diego, Calif.), and transfected into competent Escherichia coli cells. DNA from selected clones was prepared and sequenced using the Sequenase version 2 kit (U.S. Biochemicals, Cleveland, Ohio). PCR of the env TM cytoplasmic tail was also performed using the outside primers listed above on genomic DNA from cultures in which cell-free viruses had been serially passaged six times in CEMx174 cells.
Competition studies of viral fitness. (i) Preparation of infected-cell and virus stocks. Infectious virions were produced by calcium phosphate transfection of HEK293T cells. The 5' and 3' hemigenomes of SIVmac239 (or specific mutant derivatives) were linearized with SphI, purified by phenol-chloroform extraction, and resuspended in water prior to transfection (50). At 24 h posttransfection, the supernatant was clarified by centrifugation, filtered through a 0.2-µm-pore-size filter, and incubated with 4 × 106 CEMx174 cells in the presence of Polybrene (8 µg/ml) for 6 h at 37°C. The cultures were washed once with phosphate-buffered saline and maintained by adding fresh cells and/or medium as appropriate.
(ii) Fitness assay.
For the competitions between SIVmac239
and SIVmac239GY, infected CEMx174 cultures were established
with the respective virus strains. Different ratios of infected cells
from each culture were mixed with uninfected CEMx174 cells, and
uininfected cells were added daily. For analysis of DNA,
106 cells were lysed in 500 µl of PCR lysis buffer
(0.001% sodium dodecyl sulfate, 0.001% Triton X-100, 10 mM Tris-HCl
[pH 8.0], 1 mM EDTA) with proteinase K (1 mg/ml), incubated at 58°C
for 1 h, and heat inactivated at 95°C for 10 min. A 25-µl
sample was for PCR amplification of env regions with primers
oJD11 and oJD13 (sequences are given in Table
1). A 5-µl volume of reaction products was analyzed on a 3.5% MetaPhor agarose gel. For fluorescence resonance energy transfer (FRET) analysis, the cell lysates were further cleaned by phenol-chloroform extraction and resuspended in 500 µl of distilled water. Fluorescent PCR was performed with the
LightCycler (Roche Molecular Biochemicals) as specified by the
manufacturer, with FRET probes purchased from GenSet Oligos. Briefly,
the 20-µl PCR mixes contained 2 µl of cell lysate, 0.2 µM M1
probe, 0.4 µM A1 probe, 0.3 µM oJD32 primer, 0.3 µM oJD33 primer,
3 mM MgCl2, and 2 µl of DNA-Hybridization Probe Master Mix (Roche Molecular Biochemicals). The lysates were subjected to 50 cycles of amplification (95°C for 0 s, 52°C for 10 s,
72°C for 21 s) followed by melting-curve analysis. To generate
the melting curves, fluorescence was monitored continuously as the reaction temperature was increased from 43 to 95°C at a rate of 0.1°C/s.
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GY and SIVmac239Y/I and between
SIVmac239GY and SIV-7-14, infected cells, mixed cultures, and
lysates were prepared as described above. The same primers, probes, and
reaction mix were used for the SIVmac239GY-versus-SIVmac239Y/I competition. For SIVmac239GY versus SIV-7-14, the M1 and A1
probes were replaced with A2 and M2 probes, respectively, and 0.32 µl of the TaqStart anti-Taq polymerase antibody (Sigma) was
added. For this competition the amplification parameters were altered (95°C for 0 s, 56°C for 5 s, and 72°C for 21 s).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Derivation and characterization of SIVs with mutations in the
Yxx sorting motif.
To evaluate the effects of the conserved
Yxx motif in an in vivo model, we constructed two mutants using a
pathogenic molecular clone SIVmac239 (46) (Fig. 1). In one
mutant, designated SIVmac239Y/I, the critical Tyr in the motif
(YRPV) was changed to an isoleucine (Ile) by mutating two
nucleotides in env codon 721 (TAT
ATT). In a second
mutant, designated SIVmac239GY, the Tyr and the preceding glycine
(Gly) were removed by deleting the six nucleotides coding for these
amino acids. Previous studies from our laboratory (9) and
by Boge et al. (8) have shown that both Gly-720 and
Tyr-721 are critical for the formation of a functional endocytosis
signal. To monitor the replication competence of these viruses in vivo, we elected to use as the parental virus a SIVmac239 clone that contained a stop codon in nef at position 92. As shown
previously, this premature termination codon characteristically reverts
to an open reading frame within 2 to 4 weeks of inoculation of rhesus macaques, and within 1 year the majority of infected animals develop high viral loads, decreased numbers of CD4+ cells, and
clinical AIDS (46). As a result of the rapid reversion of
this nef point mutation, the clinical course of animals
infected with this clone and the course of animals infected with
SIVmac239 with an intact nef open reading frame are
identical (46).
GY was
compared to that of wild-type SIVmac239 in CEMx174 cells and PBMC
from rhesus macaques (Fig. 2). Both
mutants were infectious and yielded growth curves similar to that of
wild-type SIVmac239. The stability of the mutations during in vitro
propagation was also determined by multiple serial passages of these
viruses in CEMx174 cells, followed by amplification and sequencing of
env genes from genomic DNA. After as many as six passages in
CEMx174 cells, the mutations introduced into SIVmac239Y/I and
SIVmac239GY remained intact and no new mutations were observed in
the membrane-spanning domain or elsewhere in the TM cytoplasmic tail
(data not shown). Thus, mutations that abrogated this Tyr-based sorting
signal were stable in vitro and resulted in viruses that were
replication competent in macaque PBMC and a human lymphoid cell line.
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Inoculation of rhesus macaques with Tyr mutants.
The virus
stocks of the wild-type and mutant SIVs generated by electroporation of
CEMx174 cells were used to inoculate rhesus macaques intravenously. Two
animals received SIVmac239Y/I, three received SIVmac239GY, and three
received wild-type SIVmac239. After inoculation, plasma viral RNA
levels, percentages of CD4+ T cells, platelet counts, and
antibody responses to viral antigens were monitored every 2 to 4 weeks.
Among the three animals (K7K, N7K, and N2V) that received wild-type
SIVmac239 containing the Nef point mutation, viral RNA levels in plasma
peaked between 1.7 × 105 and 2.7 × 107 copies/ml during the first month of infection (Fig.
3). This acute phase was followed by
establishment of viral "set points" that ranged between
approximately 5 × 104 and 1 × 106
RNA copies/ml. All three animals exhibited declines in CD4+
T-cell percentages and platelet counts and were euthanized at weeks 70, 73, and 81 with weight loss and clinical features of immunodeficiency
(Table 2).
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|
GY. In WD1 and AGP, peak viral
levels of 5.5 × 104 and 1.2 × 104
RNA copies/ml, respectively, were observed. Both of these animals have
remained healthy with low or undetectable levels of RNA by the bDNA or
the QC RT-PCR assay (Fig. 3) and have maintained normal hematologic
values. Interestingly, the remaining animal (2AJ) exhibited a high peak
level of 4.8 × 105 RNA copies/ml and an elevated
viral set point of approximately 2 × 105 RNA
copies/ml of plasma. This animal subsequently experienced a progressive
increase in viral load to 2.8 × 106 RNA copies/ml and
a progressive decline in CD4+ T cells and platelets and was
euthanized at week 77 due to AIDS-defining conditions. In addition to
levels of cell-free virus in plasma, the frequency with which virus is
isolated from PBMC of SIV-infected macaques can be a direct reflection
of the viral burden (41). Virus was isolated on 100% of
attempts from the three macaques infected with the wild-type SIVmac239
as well as from macaque VF3, infected with the SIVmac239Y/I mutant.
Likewise, 95.7% of virus isolation attempts were successful for
macaque 2AJ, infected with the SIVmac239GY mutant. That the last two
animals were the only two mutant-virus recipients that maintained high
plasma viral RNA levels and died of AIDS is consistent with the
frequencies of virus isolation. In contrast, virus was successfully
isolated from PBMC from the three surviving macaques in only 24, 33, and 40% of attempts.
Thus, of the five animals that received viruses with mutations in the
Yxx motif, one of two animals infected with SIVmac239Y/I and two
of three animals infected with SIVmac239GY had clinical courses
characterized by low to undetectable viral loads, stable CD4+ T-cell percentages, and no symptoms of disease.
Similar to other published reports for SIV and HIV, the viral load was
predictive of the clinical outcome (41, 47, 65, 73, 88,
101). After the initial peak of viremia, all five animals in
which viral loads ultimately stabilized at >105 viral RNA
copies/ml later developed immunodeficiency and other characteristics of
SIV-related disease.
Antibody responses in infected macaques.
It was possible that
the attenuated course of infection in the three animals that received
mutant viruses but survived resulted from differences in host humoral
immune responses. To test this hypothesis, serum antibody titers and
the ability of antibodies to neutralize SIV were measured by
enzyme-linked immunosorbent assay (ELISA) and a CEMx174 cell-killing
assay, respectively. Within 4 weeks after inoculation, antibodies to
SIV were detected in sera from all animals (Fig.
4A to C). Also within 4 weeks, all eight
macaques had antibodies capable of neutralizing H9-adapted SIVmac251;
however, until week 16 none had antibodies that neutralized the
parental SIVmac239 grown in rhesus PBMC (Fig. 4D). Without exception,
the highest titers in both the ELISA and neutralization assays were
generated in the animals that had the highest viral loads and succumbed
to disease. Thus, these results show that the failure of animals to
develop disease was not due to a more efficient overall humoral immune
response. However, despite the low to undetectable levels of plasma
viral RNA in the surviving macaques, N8G, WD1, and AGP, these animals
maintained antibody titers ranging from 1:25,600 to 1:102,400 for up to
139 weeks after inoculation, indicating ongoing viral replication (Fig. 4).
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Molecular analysis of Env.
To determine the stability of the
Y721I and GY mutations in the inoculated animals, partial
env sequences encompassing the TM membrane-spanning
domain and the entire cytoplasmic domain were amplified by nested PCR
from peripheral blood cellular DNA. In animals that received wild-type
SIVmac239, only sporadic mutations were observed in clones from PBMC
obtained 12 and 26 weeks after inoculation (Fig.
5A); no mutations
involved the YRPV signal. Mutation of an Arg to a Gly at codon 751 in
the cytoplasmic tail was observed consistently, however, indicating
selection pressure at this position. The mutation also encodes a
Lys-to-Arg substitution at codon 81 in Rev. Although the
significance of this mutation is unclear, an identical change has been
reported in other studies of SIVmac239 in rhesus macaques
(34). Nonetheless, the lack of any mutations in the YRPV
signal is consistent with the highly conserved nature of this domain
(50, 62).
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GY, viral sequences from both WD1
and AGP continued to show the GY deletion without other mutations for
up to 56 and 52 weeks, respectively. However, although the GY
mutation persisted in all clones from animal 2AJ, which developed a
high viral load and immunodeficiency, other mutations were noted,
including Y696H in the membrane-spanning domain and a S727P and R746K
in the cytoplasmic domain (Fig. 5C). Additional sporadic mutations seen
in more than three clones included P723S, T789N, and H831L (data not
shown), as well as the R751G mutation observed in other animals.
Therefore, in the two animals that received Y/I viruses, reversion of
the mutation at position 721 was associated with a high viral load and
disease while persistence of the Ile was associated with a markedly
attenuated clinical course. In the three animals that received the
GY mutant virus, all maintained the GY deletion, with two exhibiting
low viral loads. The remaining animal developed a high viral load and
disease in association with novel mutations in the TM tail, raising the
possibility that one or more of these changes could have compensated
for the GY mutation.
Evaluation of the Nef termination codon.
As noted above, to
monitor the replication competence of viruses in vivo, animals were
inoculated with SIVs that contained a premature termination codon in
nef. We reasoned that if viruses could replicate in vivo,
this codon would be repaired rapidly. Indeed, in all three control
animals that received wild-type SIVmac239, the nef stop
codon (TAA) reverted to an open reading frame by 4 to 8 weeks after
inoculation (Table 3). Codons for a
variety of amino acids were seen in this position with Glu (GAA or GAG) or Gln (CAA) predominating and Lys (AAA), Ser (TCA), Tyr (TAC), Leu
(TTA), and Asp (GAC) observed less frequently. A stop codon in one of
eight clones from animal N2V was still detectable at 8 weeks after
inoculation. In one (VF3) of the two animals that received
SIVmac239Y/I, the nef stop codon reverted to Glu, Tyr, or
Gln in 11 of 11 clones by 2 weeks. In N8G, reversion was observed in 4 of 10 clones at 4 weeks, 6 of 6 clones at 12 weeks, and 7 of 10 clones
at 80 weeks. Among the animals that received the GY mutant, 11 of 11 clones from 2AJ had reverted to an open reading frame by 2 weeks
whereas reversions were seen in clones from both WD1 and AGP at 2 and 8 weeks, respectively. By 44 and 25 weeks, all the nef clones
had reverted in these animals. Thus, although stop codons in
nef were still detected in clones from N8G at late time
points, by 2 to 4 weeks after inoculation all the animals that remained
healthy harbored viruses with clear evidence of correction of the
nef termination codon. This finding indicated that the
mutant viruses replicated sufficiently in vivo to repair nef
and, more importantly, that a loss of Nef function could not account
for the attenuated disease course in these animals.
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Evaluation of replication competence in vitro using virus fitness
assays.
Given the reduced viral RNA load in three of five animals
that received mutant viruses, it was possible that the mutations had
introduced subtle defects in viral replicative capacity that were not
detectable in the initial growth curve experiments on PBMCs or CEMx174
cells (Fig. 1). To evaluate this possibility, viruses were compared in
a series of fitness assays. Wild-type SIVmac239 and viruses containing
the Y/I or GY mutations were combined in different ratios to
establish starting mixtures for each competition and added to CEMx174
cells. Cultures were replenished with uninfected cells (usually every 2 to 3 days) and maintained for 4 to 5 weeks. Total cellular DNA was
isolated at various time points for analysis.
GY at a starting ratio of ~1:10. Serial DNA
samples were evaluated by PCR amplification of the region of
env that included the GY mutation and analyzed by agarose
gel electrophoresis. The 6-nucleotide deletion present in
SIVmac239GY yielded a fragment with higher electrophoretic
mobility than that from the wild-type virus. As shown in Fig.
6A, although SIVmac239GY was the
predominant virus at early time points, by day 23 it was substantially
replaced by wild-type viruses and by day 35 only SIVmac239 could be
detected. No reversion of the GY mutation was observed when
SIVmac239GY was passaged alone (data not shown). Therefore, in the
context of this fitness assay, the GY mutation did introduce a
replication defect in SIVmac239.
|
GY (exhibiting a lower Tm) increased
over time until only SIVmac239 was detected on day 35 (Fig. 6B). Thus,
this result corresponded precisely to that determined by gel analysis and validated the use of this technique for subsequent fitness assays.
Experiments were next performed on a derivative of
SIVmac239GY, designated 7-14, that contained the Y696H,
P723S, S727P, R746K, R751G, T789N, and H831L mutations. These mutations
had been noted in several env clones from 2AJ, the
SIVmac239GY-inoculated animal that developed a high virus load and
disease (Fig. 5 and data not shown). Interestingly, in fitness assays,
7-14 possessed a clear replication advantage over SIVmac239GY when
these viruses were mixed at an initial ratio of ~1:3 (Fig. 6C).
Therefore, some or all of the env mutations that were
acquired in vivo in 2AJ were able to improve the replication competence
of SIVmac239GY in vitro.
In contrast to SIVmac239GY, no differences were observed in fitness
assays when SIVmac239Y/I was mixed with wild-type SIVmac239. When
approximately a 3:1 ratio of SIVmac239Y/I to SIVmac239 was added, the
same proportion was present after 28 days (Fig. 6D). Similar results
were seen over a range of input ratios (data not shown). Thus, although
striking in vivo differences were observed between animals N8G, in
which the Y/I mutation was maintained, and VF3, in which the Ile
reverted to a Tyr, no differences in replication between SIVmac239Y/I
and wild-type SIVmac239 were apparent in this in vitro fitness assay.
| |
DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
In this study we show that mutation of a highly conserved sorting
motif in the cytoplasmic tail of the SIV Env protein results in marked
in vivo attenuation of a pathogenic molecular clone. Among five animals
inoculated with viruses containing mutations that abrogated the
function of this motif, three of them exhibited an initial peak of
viremia followed by a profound reduction in viral load and have
remained healthy for up to 140 weeks. In one (N8G) of two animals that
received SIVmac239Y/I, the Y721I mutation was maintained and was
associated with an attenuated phenotype, while in the other animal
(VF3) the Ile reverted to a Tyr and was associated with a high viral
load, loss of CD4+ cells, and disease. Similarly, two (AGP
and WD1) of the three animals that received SIVmac239GY remained
healthy, with low to undetectable plasma viral RNA levels. The third
animal (2AJ) developed high viral RNA levels and disease and exhibited
novel and possibly compensatory mutations that flanked the GY
mutation. The replication competence of SIVmac239Y/I and SIVmac239GY
was demonstrated in cultured PBMC and CEMx174 cells. Moreover, these viruses produced initial peaks of viremia in all animals and corrected a premature stop codon in nef that has been shown to revert
in the setting of active in vivo but not in vitro replication
(49). Although virus fitness experiments demonstrated a
replication defect for SIVmac239GY compared to SIVmac239, no
defect was demonstrated for viruses with a Y/I mutation. In addition,
although viral RNA levels remained low or undetectable in the three
animals showing an attenuated clinical course, all have maintained
antibody titers of >1:10,000, indicating ongoing replication of the
mutant viruses. These findings demonstrate that the Tyr-dependent Env
sorting motif, while dispensable for SIV replication in vitro, has a
marked effect on viral pathogenesis.
Several attenuated SIVs that establish low viral loads and exhibit reduced pathogenicity in infected animals have been described (30, 44, 46, 57, 63, 102). However, even when the determinants for this phenotype were mapped to particular genes, such as nef, the underlying mechanisms remained unclear (23, 75, 91). In most SIV models, the risk of developing disease can be predicted by plasma viral RNA levels (41, 47, 88, 97, 101). Therefore, any intrinsic defect in virus replication could result in lower viral loads and decreased pathogenicity. Alternatively, attenuation in vivo could result from reduced cytopathicity of virus for infected cells, an inability of the virus to infect critical target sites, and/or an increased susceptibility of the virus to host immune responses. For the nef-deleted viruses, Nef downregulates major histocompatibility complex class I molecules, thereby reducing the susceptibility of infected cells to killing by virus-specific cytotoxic T lymphocytes (CTL) (17-19). Given that animals infected with nef-deleted viruses exhibit potent antiviral CTL activity, it is tempting to speculate that these viruses are less fit to survive a host cellular immune response (38, 43). However, Nef also binds cellular kinases (11, 58, 83) and increases viral infectivity (14, 26, 58, 67, 90), and as a result, nef-deleted viruses could exhibit multiple defects to account for their reduced virulence.
Although the viral env gene mediates functions that are crucial for infectivity, including binding to CD4 and a chemokine receptor and fusion with the cell membrane, there are few reports of mutations in distinct functional domains of Env that reduce pathogenicity in vivo. Reitter et al. have recently shown that SIVmac239 mutants lacking glycosylation sites in the V1/V2 loops of gp120 were markedly attenuated in vivo, possibly as a result of their ability to elicit novel humoral immune responses (76). Interestingly, in SIVs with prematurely truncated cytoplasmic tails, strong selection pressures in vivo restore a full-length TM (49). For one well-characterized attenuated isolate, SIVmac1A11, reversion of a truncated to a full-length cytoplasmic tail correlated with increases in viral load (57). A recent study by Shacklett et al. showed that multiple stop codons in the TM cytoplasmic tail of SIVmac239 reduced the replicative capacity in vitro and produced a highly attenuated phenotype in vivo (86). In addition to the Tyr-based motif evaluated in our study, the TM tail of SIV and HIV contains other domains that have been implicated in mediating cytopathic effects (95, 96) and in interacting with the viral matrix protein (20, 70). Thus, the cytoplasmic domain is likely to play an important role in virus assembly, replication, and pathogenesis (7, 31, 35, 36, 71), and in this regard our findings draw particular attention to the importance of this conserved Tyr-sorting signal.
A number of mechanisms could account for the striking in vivo effects
of mutations in this TM sorting motif. As noted previously, this domain
functions as a potent endocytosis signal that mediates clathrin-dependent internalization of Env molecules (7, 8, 50,
74, 78, 82). As a result, Env glycoproteins that are not
incorporated into budding virions are rapidly internalized and cleared
from the plasma membrane (32). SIVs and HIVs lacking this
motif characteristically exhibit a higher steady-state level of Env on
the cell surface (27, 50, 82) and could be more susceptible to humoral immune responses (7, 27, 62).
Indeed, we have shown that cells infected by SIVmac239Y/I
characteristically show three to four times more Env on the cell
surface than do cells infected by wild-type SIVmac239
(50), and we have seen a similar increase in Env
production in cells infected by SIVmac239GY (J. A. Hoxie,
unpublished data). Although this latter effect is modest, it is
possible that even a slight increase in the susceptibility of
virus-producing cells to host immune responses could have significant consequences on net virus production since this defect is amplified through subsequent rounds of infection. Alternatively, the ability of
this domain to also function as a basolateral sorting signal could be
important in directing virus infection to crucial target sites, such as
gut-associated lymphoid tissue, where viral replication is particularly
active in early stages of infection (99). Finally, it is
also possible that the loss of a determinant for directional virus
spread could impact on the efficiency of cell-to-cell transmission in
tissues. Indeed, the loss of the corresponding Yxx signal in an
HIV-1 TM recently was shown to be associated with a defect in
cell-to-cell spread in vitro (27).
It remains possible that the subtle defect in the replicative capacity
of SIVmac239GY contributed to its attenuated in vivo phenotype.
Although intrinsic differences in infection kinetics compared to those
of wild-type SIVmac239 were not seen in growth curves generated with
PBMC or CEMx174 cells, SIVmac239GY was less fit in competition
assays. The slightly lower levels of plasma RNA reached during the
acute phase of infection in two of three animals infected with
SIVmac239GY are consistent with this observation. However, for
SIVmac239Y/I, no differences were detectable with respect to SIVmac239
in fitness assays, although viruses in the one animal that maintained
this mutation exhibited an attenuated phenotype. Although a selection
pressure to restore the Tyr was evident in animal VF3, the finding that
this mutation did not revert in vitro during multiple passages
(50; Hoxie, unpublished) indicates that elimination
of this sorting motif has little or no effect on virus replication, at
least under these conditions. Finally, as noted above, reversion of the
nef premature termination codon was observed in all animals,
indicating that there was a sufficient level of replication to restore
this open reading frame. Although it is possible that the attenuating
effect of mutations in the Yxx motif is context dependent and
applicable only for viruses containing a stop codon in nef,
we consider this possibility unlikely, given that reversion to a
nef open reading frame was observed in all animals within 8 weeks after inoculation. Our data are consistent with the view that the
Yxx sorting motif in SIV is a crucial determinant of virus
replication in vivo and/or in modulating susceptibility of
virus-producing cells to host immune responses.
It is interesting that animal 2AJ developed a high viral load despite
the continued presence of the GY mutation. Although it is possible
that additional mutations in the 2AJ cytoplasmic domain compensated for
this effect, none of the mutations in clones from this animal created
an alternate Yxx motif. However, it remains possible that one or
more of these new mutations facilitated an interaction with cellular
adapter proteins or other molecules to generate a novel endocytosis
and/or basolateral sorting signal. Alternatively these changes could
have affected other parts of the viral life cycle, a finding suggested
by the improved fitness of a virus that contained the GY mutation
and the compensatory mutations in a 2AJ clone (Fig. 6C). Additional in
vivo studies are required to determine if one or more of these
mutations is sufficient to account for the aggressive disease course
that was observed in this animal.
Attenuated SIVs have enabled determinants for pathogenesis to be identified that could not have been inferred from in vitro studies. Moreover, as shown by the occurrence of nef-deleted HIV-1s that are associated with either absent or delayed progression to disease, these animal models will probably have relevance for HIV infection (25, 48, 51). Importantly, attenuated SIV models have provided the best evidence to date that protective immune responses can be generated, and intensive efforts are being undertaken to understand the basis for this effect (3, 28, 42, 52). The use of an attenuated SIV or HIV vaccine poses obvious safety concerns and has prompted the development of second- and third-generation viruses with multiple defects that impair replication (44). However, while increasingly attenuated viruses may be safer, theoretically there appear to be "threshold effects" in that viruses that are more severely impaired in their ability to replicate in vivo have not been able to generate protective immunity (44, 64, 80, 98). Approaches that reduce viral pathogenicity while preserving an immune response would be highly desirable, and in this regard, strategies that alter the presentation of viral proteins to the host immune system could be advantageous. Our findings indicate that the SIV TM cytoplasmic tail, and in particular this Tyr-based sorting motif, can be an additional target for attenuation strategies. Additional studies to determine the mechanism of this effect, its impact on the host immune response, and the ability of Tyr-721 mutants to generate protective immunity will be of considerable interest.
| |
ACKNOWLEDGMENTS |
|---|
We thank Peter Daly (Chiron Corp.) for performing bDNA assays and Beth Haggarty, Jackie Stallworth, Teresa Wiltrout, and Pamela May for expert technical assistance.
This work was supported by PHS grants RO1 AI33854 (J.A.H.), AI32377 (P.N.F.), AI46246 (M.H.M.), and P30-AI27767 for the flow cytometry core of the UAB Center for AIDS Research, a grant from the United Kingdom Medical Research Council (M.M.), and funds from the National Cancer Institute, National Institutes of Health, under contract NO1-CO-56000. M.M. and J.A.H. were also supported by a NATO collaboration grant. J.D.D. was supported by NIH Medical Scientist Training Program grant GM07170.
| |
FOOTNOTES |
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* Corresponding author. Mailing address: Rm. 356, Biomedical Research Building II/III, University of Pennsylvania, 421 Curie Blvd., Philadelphia, PA 19104. Phone: (215) 898-0261. Fax: (215) 573-7356. E-mail: hoxie{at}mail.med.upenn.edu.
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REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. | Aiken, C., J. Konner, N. R. Landau, M. E. Lenburg, and D. Trono. 1994. Nef induces CD4 endocytosis: requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain. Cell 76:853-864[CrossRef][Medline]. |
| 2. |
Alexander, L.,
E. Weiskopf,
T. C. Greenough,
N. C. Gaddis,
M. R. Auerbach,
M. H. Malim,
S. J. O'Brien,
B. D. Walker,
J. L. Sullivan, and R. C. Desrosiers.
2000.
Unusual polymorphisms in human immunodeficiency virus type 1 associated with nonprogressive infection.
J. Virol.
74:4361-4376 |
| 3. |
Almond, N., and J. Stott.
1999.
Live attenuated SIV a model of a vaccine for AIDS.
Immunol. Lett.
66:167-170[CrossRef][Medline].
|
| 4. |
Baba, T. W.,
Y. S. Jeong,
D. Pennick,
R. Bronson,
M. F. Greene, and R. M. Ruprecht.
1995.
Pathogenicity of live, attenuated SIV after mucosal infection of neonatal macaques.
Science
267:1820-1825 |
| 5. | Baba, T. W., V. Liska, A. H. Khimani, N. B. Ray, P. J. Dailey, D. Penninck, R. Bronson, M. F. Greene, H. M. McClure, L. N. Martin, and R. M. Ruprecht. 1999. Live attenuated, multiply deleted simian immunodeficiency virus causes AIDS in infant and adult macaques. Nat. Med. 5:194-203[CrossRef][Medline]. |
| 6. | Baur, A. S., E. T. Sawai, P. Dazin, W. J. Fantl, C. Cheng-Mayer, and B. M. Peterlin. 1994. HIV-1 Nef leads to inhibition or activation of T cells depending on its intracellular localization. Immunity 1:373-384[CrossRef][Medline]. |
| 7. |
Berlioz-Torrent, C.,
B. L. Shacklett,
L. Erdtmann,
L. Delamarre,
I. Bouchaert,
P. Sonigo,
M. C. Dokhelar, and R. Benarous.
1999.
Interactions of the cytoplasmic domains of human and simian retroviral transmembrane proteins with components of the clathrin adapter complexes modulate intracellular and cell surface expression of envelope glycoproteins.
J. Virol.
73:1350-1361 |
| 8. |
Boge, M.,
S. Wyss,
J. S. Bonifacino, and M. Thali.
1998.
A membrane-proximal tyrosine-based signal mediates internalization of the HIV-1 envelope glycoprotein via interaction with the AP-2 clathrin adapter.
J. Biol. Chem.
273:15773-15778 |
| 9. | Bowers, K., A. Pelchen-Matthews, S. Höning, P. J. Vance, L. Creary, B. S. Haggarty, J. Romano, W. Ballensiefen, J. A. Hoxie, and M. Marsh. 2000. The simian immunodeficiency virus envelope glycoprotein contains multiple signals that regulate its cell surface expression and endocytosis. Traf 1:661-674. |
| 10. | Brander, C., and B. D. Walker. 1999. T lymphocyte responses in HIV-1 infection: implications for vaccine development. Curr. Opin. Immunol. 11:451-459[CrossRef][Medline]. |
| 11. |
Brown, A.,
X. Wang,
E. Sawai, and C. Cheng-Mayer.
1999.
Activation of the PAK-related kinase by human immunodeficiency virus type 1 Nef in primary human peripheral blood lymphocytes and macrophages leads to phosphorylation of a PIX-p95 complex.
J. Virol.
73:9899-9907 |
| 12. |
Carrington, M.,
M. Dean,
M. P. Martin, and S. J. O'Brien.
1999.
Genetics of HIV-1 infection: chemokine receptor CCR5 polymorphism and its consequences.
Hum. Mol. Genet.
8:1939-1945 |
| 13. |
Carrington, M.,
G. W. Nelson,
M. P. Martin,
T. Kissner,
D. Vlahov,
J. J. Goedert,
R. Kaslow,
S. Buchbinder,
K. Hoots, and S. J. O'Brien.
1999.
HLA and HIV-1: heterozygote advantage and B*35-Cw*04 disadvantage.
Science
283:1748-1752 |
| 14. |
Chowers, M. Y.,
C. A. Spina,
T. J. Kwoh,
N. J. Fitch,
D. D. Richman, and J. C. Guatelli.
1994.
Optimal infectivity in vitro of human immunodeficiency virus type 1 requires an intact nef gene.
J. Virol.
68:2906-2914 |
| 15. | Clements, J. E., R. C. Montelaro, M. C. Zink, A. M. Amedee, S. Miller, A. M. Trichel, B. Jagerski, D. Hauer, L. N. Martin, and R. P. Bohm. 1995. Cross-protective immune responses induced in rhesus macaques by immunization with attenuated macrophage-tropic simian immunodeficiency virus. J. Virol. 69:2737-2744[Abstract]. |
| 16. | Coffin, J. M. 1995. HIV population dynamics in vivo: implications for genetic variation, pathogenesis, and therapy. Science 267:483-489. |
| 17. | Cohen, G. B., R. T. Gandhi, D. M. Davis, O. Mandelboim, B. K. Chen, J. L. Strominger, and D. Baltimore. 1999. The selective downregulation of class I major histocompatibility complex proteins by HIV-1 protects HIV-infected cells from NK cells. Immunity 10:661-671[CrossRef][Medline]. |
| 18. | Collins, K. L., and D. Baltimore. 1999. HIV's evasion of the cellular immune response. Immunol. Rev. 168:65-74[CrossRef][Medline]. |
| 19. | Collins, K. L., B. K. Chen, S. A. Kalams, B. D. Walker, and D. Baltimore. 1998. HIV-1 Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes. Nature (London) 391:397-401[CrossRef][Medline]. |
| 20. | Cosson, P. 1996. Direct interaction between the envelope and matrix. EMBO J. 15:5783-5788[Medline]. |
| 21. |
Craig, H. M.,
M. W. Pandori, and J. C. Guatelli.
1998.
Interaction of HIV-1 Nef with the cellular dileucine-based sorting pathway is required for CD4 down-regulation and optimal viral infectivity.
Proc. Natl. Acad. Sci. USA
95:11229-11234 |
| 22. | Cranage, M. P., A. M. Whatmore, S. A. Sharpe, N. Cook, N. Polyanskaya, S. Leech, J. D. Smith, E. W. Rud, M. J. Dennis, and G. A. Hall. 1997. Macaques infected with live attenuated SIVmac are protected against superinfection via the rectal mucosa. Virology 229:143-154[CrossRef][Medline]. |
| 23. | Cullen, B. R. 1998. HIV-1 auxiliary proteins: making connections in a dying cell. Cell 93:685-692[CrossRef][Medline]. |
| 24. |
Daniel, M. D.,
F. Kirchhoff,
S. C. Czajak,
P. K. Sehgal, and R. C. Desrosiers.
1992.
Protective effects of a live attenuated SIV vaccine with a deletion in the nef gene.
Science
258:1938-1941 |
| 25. |
Deacon, N. J.,
A. Tsykin,
A. Solomon,
K. Smith,
M. Ludford-Menting,
D. J. Hooker,
D. A. McPhee,
A. L. Greenway,
A. Ellett, and C. Chatfield.
1995.
Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients.
Science
270:988-991 |
| 26. | de Ronde, A., B. Klaver, W. Keulen, L. Smit, and J. Goudsmit. 1992. Natural HIV-1 NEF accelerates virus replication in primary human lymphocytes. Virology 188:391-395[CrossRef][Medline]. |
| 27. |
Deschambeault, J.,
J. P. Lalonde,
G. Cervantes-Acosta,
R. Lodge,
E. A. Cohen, and G. LeMay.
1999.
Polarized human immunodeficiency virus budding in lymphocytes involves a tyrosine-based signal and favors cell-to-cell viral transmission.
J. Virol.
73:5010-5017 |
| 28. | Desrosiers, R. C. 1998. Prospects for live attenuated HIV. Nat. Med. 4:982[Medline]. |
| 29. | Desrosiers, R. C. 1999. Strategies used by human immunodeficiency virus that allow persistent viral replication. Nat. Med. 5:723-725[CrossRef][Medline]. |
| 30. |
Desrosiers, R. C.,
J. D. Lifson,
J. S. Gibbs,
S. C. Czajak,
A. Y. Howe,
L. O. Arthur, and R. P. Johnson.
1998.
Identification of highly attenuated mutants of simian immunodeficiency virus.
J. Virol.
72:1431-1437 |
| 31. |
Dubay, J. W.,
S. J. Roberts,
B. H. Hahn, and E. Hunter.
1992.
Truncation of the human immunodeficiency virus type 1 transmembrane glycoprotein cytoplasmic domain blocks virus infectivity.
J. Virol.
66:6616-6625 |
| 32. |
Egan, M. A.,
L. M. Carruth,
J. F. Rowell,
X. Yu, and R. F. Siliciano.
1996.
Human immunodeficiency virus type 1 envelope protein endocytosis mediated by a highly conserved intrinsic internalization signal in the cytoplasmic domain of gp41 is suppressed in the presence of the Pr55gag precursor protein.
J. Virol.
70:6547-6556 |
| 33. |
Fauci, A. S.
1999.
The AIDS epidemicconsiderations for the 21st century.
N. Engl. J. Med.
341:1046-1050 |
| 34. | Flaherty, M. T., D. A. Hauer, J. L. Mankowski, M. C. Zink, and J. E. Clements. 1997. Molecular and biological characterization of a neurovirulent molecular clone of simian immunodeficiency virus. J. Virol. 71:5790-5798[Abstract]. |
| 35. | Freed, E. O., and M. A. Martin. 1996. Domains of the human immunodeficiency virus type 1 matrix and gp41 cytoplasmic tail required for envelope incorporation into virions. J. Virol. 70:341-351[Abstract]. |
| 36. |
Gabuzda, D. H.,
A. Lever,
E. Terwilliger, and J. Sodroski.
1992.
Effects of deletions in the cytoplasmic domain on biological functions of human immunodeficiency virus type 1 envelope glycoproteins.
J. Virol.
66:3306-3315 |
| 37. | Garcia, J. V., and A. D. Miller. 1991. Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature (London) 350:508-511[CrossRef][Medline]. |
| 38. | Gauduin, M. C., R. L. Glickman, S. Ahmad, T. Yilma, and R. P. Johnson. 1999. Characterization of SIV-specific CD4+ T-helper proliferative responses in macaques immunized with live-attenuated SIV. J. Med. Primatol. 28:233-241[Medline]. |
| 39. | Greenberg, M. E., A. J. Iafrate, and J. Skowronski. 1998. The SH3 domain-binding surface and an acidic motif in HIV-1 Nef regulate trafficking of class I MHC complexes. EMBO J. 17:2777-2789[CrossRef][Medline]. |
| 40. | Hanna, Z., D. G. Kay, N. Rebai, A. Guimond, S. Jothy, and P. Jolicoeur. 1998. Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice. Cell 95:163-175[CrossRef][Medline]. |
| 41. |
Hirsch, V. M.,
G. Dapolito,
A. Hahn,
J. Lifson,
D. Montefiori,
C. R. Brown, and R. Goeken.
1998.
Viral genetic evolution in macaques infected with molecularly cloned simian immunodeficiency virus correlates with the extent of persistent viremia.
J. Virol.
72:6482-6489 |
| 42. | Johnson, R. P., and R. C. Desrosiers. 1998. Protective immunity induced by live attenuated simian immunodeficiency virus. Curr. Opin. Immunol. 10:436-443[CrossRef][Medline]. |
| 43. | Johnson, R. P., R. L. Glickman, J. Q. Yang, A. Kaur, J. T. Dion, M. J. Mulligan, and R. C. Desrosiers. 1997. Induction of vigorous cytotoxic T-lymphocyte responses by live attenuated simian immunodeficiency virus. J. Virol. 71:7711-7718[Abstract]. |
| 44. |
Johnson, R. P.,
J. D. Lifson,
S. C. Czajak,
K. S. Cole,
K. H. Manson,
R. Glickman,
J. Yang,
D. C. Montefiori,
R. Montelaro,
M. S. Wyand, and R. C. Desrosiers.
1999.
Highly attenuated vaccine strains of simian immunodeficiency virus protect against vaginal challenge: inverse relationship of degree of protection with level of attenuation.
J. Virol.
73:4952-4961 |
| 45. |
Kestler, H.,
T. Kodama,
D. Ringler,
M. Marthas,
N. Pedersen,
A. Lackner,
D. Regier,
P. Sehgal,
M. Daniel,
N. King, and R. Desrosiers.
1990.
Induction of AIDS in rhesus monkeys by molecularly cloned simian immunodeficiency virus.
Science
248:1109-1112 |
| 46. | Kestler, H. W., III, D. J. Ringler, M. Kazayasu, D. L. Panicali, P. K. Sehgal, M. D. Daniel, and R. C. Desrosiers. 1991. Importance of the nef gene for maintenance of high virus loads and for development of AIDS. Cell 65:651-662[CrossRef][Medline]. |
| 47. | Kimata, J. T., L. Kuller, D. B. Anderson, P. Dailey, and J. Overbaugh. 1999. Emerging cytopathic and antigenic simian immunodeficiency virus variants influence AIDS progression. Nat. Med. 5:535-541[CrossRef][Medline]. |
| 48. |
Kirchhoff, F.,
T. C. Greenough,
D. B. Brettler,
J. L. Sullivan, and R. C. Desrosiers.
1995.
Brief report: absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection.
N. Engl. J. Med.
332:228-232 |
| 49. |
Kodama, T.,
D. P. Wooley,
Y. M. Naidu,
H. W. Kestler III,
M. D. Daniel,
Y. Li, and R. C. Desrosiers.
1989.
Significance of premature stop codons in env of simian immunodeficiency virus.
J. Virol.
63:4709-4714 |
| 50. | LaBranche, C. C., M. M. Sauter, B. S. Haggarty, P. J. Vance, J. Romano, T. K. Hart, P. J. Bugelski, M. Marsh, and J. A. Hoxie. 1995. A single amino acid change in the cytoplasmic domain of SIVmac transmembrane molecule increases envelope glycoprotein expression on cells and virions. J. Virol. 69:5217-5227[Abstract]. |
| 51. |
Learmont, J. C.,
A. F. Geczy,
J. Mills,
L. J. Ashton,
C. H. Raynes-Greenow,
R. J. Garsia,
W. B. Dyer,
L. McIntyre,
R. B. Oelrichs,
D. I. Rhodes,
N. J. Deacon, and J. S. Sullivan.
1999.
Immunologic and virologic status after 14 to 18 years of infection with an attenuated strain of HIV-1. A report from the Sydney Blood Bank Cohort.
N. Engl. J. Med.
340:1715-1722 |
| 52. |
Letvin, N. L.
1998.
Progress in the development of an HIV-1 vaccine.
Science
280:1875-1880 |
| 53. | Levy, J. A. 1996. The value of primate models for studying human immunodeficiency virus pathogenesis. J. Med. Primatol. 25:163-174[Medline]. |
| 54. |
Lodge, R.,
H. Göttlinger,
D. Gabuzda,
E. A. Cohen, and G. LeMay.
1994.
The intracytoplasmic domain of gp41 mediated polarized budding of human immunodeficiency virus type 1 in MDCK cells.
J. Virol.
68:4857-4861 |
| 55. | Lodge, R., J. P. Lalonde, G. LeMay, and E. A. Cohen. 1997. The membrane-proximal intracytoplasmic tyrosine residue of HIV-1 envelope glycoprotein is critical for basolateral targeting of viral budding in MDCK cells. EMBO J. 16:695-705[CrossRef][Medline]. |
| 56. |
Lohman, B. L.,
M. B. McChesney,
C. J. Miller,
E. McGowan,
S. M. Joye,
K. K. VanRompay,
E. Reay,
L. Antipa,
N. C. Pedersen, and M. L. Marthas.
1994.
A partially attenuated simian immunodeficiency virus induces host immunity that correlates with resistance to pathogenic virus challenge.
J. Virol.
68:7021-7029 |
| 57. | Luciw, P. A., K. E. Shaw, B. L. Shacklett, and M. L. Marthas. 1998. Importance of the intracytoplasmic domain of the simian immunodeficiency virus (SIV) envelope glycoprotein for pathogenesis. Virology 252:9-16[CrossRef][Medline]. |
| 58. | Luo, T., R. A. Livingston, and J. V. Garcia. 1997. Infectivity enhancement by human immunodeficiency virus type 1 Nef is independent of its association with a cellular serine/threonine kinase. J. Virol. 71:9524-9530[Abstract]. |
| 59. |
Mangasarian, A.,
V. Piguet,
J. K. Wang,
Y. L. Chen, and D. Trono.
1999.
Nef-induced CD4 and major histocompatibility complex class I (MHC-I) down-regulation are governed by distinct determinants: N-terminal alpha helix and proline repeat of Nef selectively regulate MHC-I trafficking.
J. Virol.
73:1964-1973 |
| 60. | Mariani, R., F. Kirchhoff, T. C. Greenough, J. L. Sullivan, R. C. Desrosiers, and J. Skowronski. 1996. High frequency of defective nef alleles in a long-term survivor with nonprogressive human immunodeficiency virus type 1 infection. J. Virol. 70:7752-7764[Abstract]. |
| 61. |
Marsh, M., and H. T. McMahon.
1999.
The structural era of endocytosis.
Science
285:215-220 |
| 62. | Marsh, M., A. Pelchen-Matthews, and J. A. Hoxie. 1997. Endocytosis and lentiviral pathogenesis. Trends Biochem. Sci. 7:1-4. |
| 63. |
Marthas, M. L.,
R. A. Ramos,
B. L. Lohman,
K. K. Van Rompay,
R. E. Unger,
C. J. Miller,
B. Banapour,
N. C. Pedersen, and P. A. Luciw.
1993.
Viral determinants of simian immunodeficiency virus (SIV) virulence in rhesus macaques assessed by using attenuated and pathogenic molecular clones of SIVmac.
J. Virol.
67:6047-6055 |
| 64. |
Marthas, M. L.,
S. Sutjipto,
J. Higgins,
B. Lohman,
J. Torten,
P. A. Luciw,
P. A. Marx, and N. C. Pedersen.
1990.
Immunization with a live, attenuated simian immunodeficiency virus (SIV) prevents early disease but not infection in rhesus macaques challenged with pathogenic SIV.
J. Virol.
64:3694 |
| 65. | Mellors, J. W., C. R. Rinaldo, Jr., P. Gupta, R. M. White, J. A. Todd, and L. A. Kingsley. 1996. Prognosis in HIV-1 infection predicted by the quantity of virus in plasma. Science 272:1167-1170[Abstract]. |
| 66. | Miller, C. J., M. B. McChesney, X. Lu, P. J. Dailey, C. Chutkowski, D. Lu, P. Brosio, B. Roberts, and Y. Lu. 1997. Rhesus macaques previously infected with simian/human immunodeficiency virus are protected from vaginal challenge with pathogenic SIVmac239. J. Virol. 71:1911-1921[Abstract]. |
| 67. |
Miller, M. D.,
M. T. Warmerdam,
I. Gaston,
W. C. Greene, and M. B. Feinberg.
1994.
The human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages.
J. Exp. Med.
179:101-113 |
| 68. | Montefiori, D. C., T. W. Baba, A. Li, M. Bilska, and R. M. Ruprecht. 1996. Neutralizing and infection-enhancing antibody responses do not correlate with the differential pathogenicity of SIVmac239delta3 in adult and infant rhesus monkeys. J. Immunol. 157:5528-5535[Abstract]. |
| 69. |
Mukherjee, S.,
R. N. Ghosh, and F. R. Maxfield.
1997.
Endocytosis.
Physiol. Rev.
77:759-803 |
| 70. |
Murakami, T., and E. O. Freed.
2000.
Genetic evidence for an interaction between human immunodeficiency virus type 1 matrix and alpha-helix 2 of the gp41 cytoplasmic tail.
J. Virol.
74:3548-3554 |
| 71. |
Murakami, T., and E. O. Freed.
2000.
The long cytoplasmic tail of gp41 is required in a cell type-dependent manner for HIV-1 envelope glycoprotein incorporation into virions.
Proc. Natl. Acad. Sci. USA
97:343-348 |
| 72. | Nilsson, C., B. Makitalo, R. Thorstensson, S. Norley, D. Binninger-Schinzel, M. Cranage, E. Rud, G. Biberfeld, and P. Putkonen. 1998. Live attenuated simian immunodeficiency virus (SIV)mac in macaques can induce protection against mucosal infection with SIVsm. AIDS 12:2261-2270[CrossRef][Medline]. |
| 73. |
O'Brien, W. A.,
P. M. Hartigan,
D. Martin,
J. Esinhart,
A. Hill,
S. Benoit,
M. Rubin,
M. S. Simberkoff, and J. D. Hamilton.
1996.
Changes in plasma HIV-1 RNA and CD4+ lymphocyte counts and the risk of progression to AIDS. Veterans Affairs Cooperative Study Group on AIDS.
N. Engl. J. Med.
334:426-431 |
| 74. | Ohno, H., R. C. Aguilar, M. C. Fournier, S. Hennecke, P. Cosson, and J. S. Bonifacino. 1997. Interaction of endocytic signals from the HIV-1 envelope glycoprotein complex with members of the adaptor medium chain family. Virology 238:305-315[CrossRef][Medline]. |
| 75. | Piguet, V., O. Schwartz, S. Le Gall, and D. Trono. 1999. The downregulation of CD4 and MHC-I by primate lentiviruses: a paradigm for the modulation of cell surface receptors. Immunol. Rev. 168:51-63[CrossRef][Medline]. |
| 76. | Reitter, J. N., R. E. Means, and R. C. Desrosiers. 1998. A role for carbohydrates in immune evasion in AIDS. Nat. Med. 4:679-684[CrossRef][Medline]. |
| 77. |
Rosenberg, E. S.,
J. M. Billingsley,
A. M. Caliendo,
S. L. Boswell,
P. E. Sax,
S. A. Kalams, and B. D. Walker.
1997.
Vigorous HIV-1-specific CD4+ T cell responses associated with control of viremia.
Science
278:1447-1450 |
| 78. | Rowell, J. F., P. E. Stanhope, and R. F. Siliciano. 1995. Endocytosis of the HIV-1 envelope protein: mechanism and role in processing for association with class II MHC. J. Immunol. 155:473-488[Abstract]. |
| 79. |
Rud, E. W.,
M. Cranage,
J. Yon,
J. Quirk,
L. Ogilvie,
N. Cook,
S. Webster,
M. Dennis, and B. E. Clarke.
1994.
Molecular and biological characterization of simian immunodeficiency virus macaque strain 32H proviral clones containing nef size variants.
J. Gen. Virol.
75:529-543 |
| 80. | Ruprecht, R. M., T. W. Baba, R. Rasmussen, Y. Hu, and P. L. Sharma. 1996. Murine and simian retrovirus models: the threshold hypothesis. AIDS 10(Suppl. A):S33-S40. |
| 81. | Samson, M., F. Libert, B. J. Doranz, J. Rucker, C. Liesnard, C. M. Farber, S. Saragosti, C. Lapoumeroulie, J. Cognaux, C. Forceille, G. Muyldermans, C. Verhofstede, G. Burtonboy, M. Georges, T. Imai, S. Rana, Y. Yi, R. J. Smyth, R. G. Collman, R. W. Doms, G. Vassart, and M. Parmentier. 1996. Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature (London) 382:722-725[CrossRef][Medline]. |
| 82. |
Sauter, M. M.,
A. Pelchen-Matthews,
R. Bron,
M. Marsh,
C. C. LaBranche,
P. J. Vance,
J. Romano,
B. S. Haggarty,
T. K. Hart,
W. M. F. Lee, and J. A. Hoxie.
1996.
An internalization signal in the simian immunodeficiency virus transmembrane protein cytoplasmic domain modulates expression of envelope glycoproteins on the cell surface.
J. Cell Biol.
132:795-811 |
| 83. |
Sawai, E. T.,
A. Baur,
H. Struble,
B. M. Peterlin,
J. A. Levy, and C. Cheng-Mayer.
1994.
Human immunodeficiency virus type 1 Nef associates with a cellular serine kinase in T lymphocytes.
Proc. Natl. Acad. Sci. USA
91:1539-1543 |
| 84. | Schmid, S. L. 1997. Clathrin-coated vesicle formation and protein sorting: an integrated process. Annu. Rev. Biochem. 66:511-548[CrossRef][Medline]. |
| 85. | Schwartz, O., V. Marechal, S. Le Gall, F. Lemonnier, and J. M. Heard. 1996. Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein. Nat. Med. 2:338-342[CrossRef][Medline]. |
| 86. |
Shacklett, B. L.,
C. J. Weber,
K. E. Shaw,
E. M. Keddie,
M. B. Gardner,
P. Sonigo, and P. A. Luciw.
2000.
The intracytoplasmic domain of the Env transmembrane protein is a locus for attenuation of simian immunodeficiency virus SIVmac in rhesus macaques.
J. Virol.
74:5836-5844 |
| 87. | Skowronski, J., D. Parks, and R. Mariani. 1993. Altered T cell activation and development in transgenic mice expressing the HIV-1 nef gene. EMBO J. 12:703-713[Medline]. |
| 88. | Smith, S. M., B. Holland, C. Russo, P. J. Dailey, P. A. Marx, and R. I. Connor. 1999. Retrospective analysis of viral load and SIV antibody responses in rhesus macaques infected with pathogenic SIV: predictive value for disease progression. AIDS Res. Hum. Retroviruses 15:1691-1701[CrossRef][Medline]. |
| 89. |
Sodora, D. L.,
K. E. Sheridan,
P. A. Marx, and R. I. Connor.
1999.
Immunization with a live, attenuated simian immunodeficiency virus vaccine leads to restriction of viral diversity in Rhesus macaques not protected from pathogenic challenge.
J. Virol.
73:4443-4446 |
| 90. |
Spina, C. A.,
T. J. Kwoh,
M. Y. Chowers,
J. C. Guatelli, and D. D. Richman.
1994.
The importance of nef in the induction of human immunodeficiency virus type 1 replication from primary quiescent CD4 lymphocytes.
J. Exp. Med.
179:115-123 |
| 91. | Stevenson, M. 1997. Molecular mechanisms for the regulation of HIV replication, persistence and latency. AIDS 11(Suppl. A):S25-S33. |
| 92. | Stott, J., S. L. Hu, and N. Almond. 1998. Candidate vaccines protect macaques against primate immunodeficiency viruses. AIDS Res. Hum. Retroviruses 14(Suppl. 3):S265-S270. |
| 93. | Suryanarayana, K., T. A. Wiltrout, G. M. Vasquez, V. M. Hirsch, and J. D. Lifson. 1998. Plasma SIV RNA viral load determination by real-time quantification of product generation in reverse transcriptase-polymerase chain reaction. AIDS Res. Hum. Retroviruses 14:183-189[Medline]. |
| 94. | Tao, B., and P. N. Fultz. 1999. Pathogenicity and comparative evolution in vivo of the transitional quasispecies SIVsmmPBj8. Virology 259:166-175[CrossRef][Medline]. |
| 95. | Tencza, S. B., T. A. Mietzner, and R. C. Montelaro. 1997. Calmodulin-binding function of LLP segments from the HIV type 1 transmembrane protein is conserved among natural sequence variants. AIDS Res. Hum. Retroviruses 13:263-269[Medline]. |
| 96. | Tencza, S. B., M. A. Miller, K. Islam, T. A. Mietzner, and R. C. Montelaro. 1995. Effect of amino acid substitutions on calmodulin binding and cytolytic properties of the LLP-1 peptide segment of human immunodeficiency virus type 1 transmembrane protein. J. Virol. 69:5199-5202[Abstract]. |
| 97. |
Ten Haaft, P.,
B. Verstrepen,
K. Uberla,
B. Rosenwirth, and J. Heeney.
1998.
A pathogenic threshold of virus load defined in simian immunodeficiency virus- or simian-human immunodeficiency virus-infected macaques.
J. Virol.
72:10281-10285 |
| 98. | Ui, M., T. Kuwata, T. Igarashi, Y. Miyazaki, K. Tamaru, T. Shimada, M. Nakamura, H. Uesaka, H. Yamamoto, and M. Hayami. 1999. Protective immunity of gene-deleted SHIVs having an HIV-1 Env against challenge infection with a gene-intact SHIV. J. Med. Primatol. 28:242-248[Medline]. |
| 99. |
Veazey, R. S.,
M. DeMaria,
L. V. Chalifoux,
D. E. Shvetz,
D. R. Pauley,
H. L. Knight,
M. Rosenzweig,
R. P. Johnson,
R. C. Desrosiers, and A. A. Lackner.
1998.
Gastrointestinal tract as a major site of CD4+ T cell depletion and viral replication in SIV infection.
Science
280:427-431 |
| 100. |
Wang, J. K.,
E. Kiyokawa,
E. Verdin, and D. Trono.
2000.
The Nef protein of HIV-1 associates with rafts and primes T cells for activation.
Proc. Natl. Acad. Sci. USA
97:394-399 |
| 101. | Watson, A., J. Ranchalis, B. Travis, J. McClure, W. Sutton, P. R. Johnson, S. L. Hu, and N. L. Haigwood. 1997. Plasma viremia in macaques infected with simian immunodeficiency virus: plasma viral load early in infection predicts survival. J. Virol. 71:284-290[Abstract]. |
| 102. | Whatmore, A. M., N. Cook, G. A. Hall, S. Sharpe, E. W. Rud, and M. P. Cranage. 1995. Repair and evolution of nef in vivo modulates simian immunodeficiency virus virulence. J. Virol. 69:5117-5123[Abstract]. |
| 103. |
Wyand, M. S.,
K. Manson,
D. C. Montefiori,
J. D. Lifson,
R. P. Johnson, and R. C. Desrosiers.
1999.
Protection by live, attenuated simian immunodeficiency virus against heterologous challenge.
J. Virol.
73:8356-8363 |
| 104. |
Xu, X. N.,
B. Laffert,
G. R. Screaton,
M. Kraft,
D. Wolf,
W. Kolanus,
J. Mongkolsapay,
A. J. McMichael, and A. S. Baur.
1999.
Induction of Fas ligand expression by HIV involves the interaction of Nef with the T cell receptor zeta chain.
J. Exp. Med.
189:1489-1496 |
Journal of Virology, January 2001, p. 278-291, Vol. 75, No. 1
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.1.278-291.2001
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